The general objective of this research is to understand the control of striated muscle contraction. The specific aims for this grant period focus on understanding the roles of CA binding and cross-bridge attachment in activating contraction. This will involve (1) testing the relative influence of each by selectively decreasing the contribution of the other, (2) studying the structural changes in the thin filament accompanying mechanical changes that affect contractile regulation, (3) testing the hypothesis that cross-bridge attachment decreases the rate of Ca dissociation from CaTn with experimental and modeling studies, (4) testing whether the hysteresis in Ca sensitivity involves the role of cross-bridges in activation or changes in lattice spacing, and (5) developing a quantitative model to explain contractile activation. In addition, specific hypotheses of the interaction of the regulatory proteins will be tested. These studies will use a wide variety of preparations from whole muscles to skinned fibers to reconstituted filaments to isolated proteins from barnacles, rabbits, frogs, and rats. Mechanical measurements will include force and stiffness. Myosin binding, and filament activation will use measurements of ATPase activity. Intracellular Ca will be measured with aequorin. Structural changes in proteins either isolated or reconstituted into filaments and fibers will be monitored by fluorescence, including steady state, FRET, or linear dichroism of extrinsic or intrinsic labels. Proteins will be either isolated as native or mutated proteins, fluorescently labeled or not, and reconstituted into filament or fibers or modified in the fiber. This will provide a more complete understanding of how muscle contraction is regulated, the central step in regulating movement, and how the movement itself affects regulation.